The challenges and prospects of capillary zone electrophoresis-mass spectrometry for characterization of monoclonal antibodies biotherapeutics

The application of Capillary Zone Electrophoresis (CZE) in proteomic analysis has increased significantly since the technique was hyphenated with high resolution mass spectrometry (HR-MS). The coupling was successful in combining the analytical features of high separation efficiency and resolving power of CZE with the precision of molecular mass measurements and sequencing of HR-MS analyzers for characterization of proteins and peptides and of their proteoforms, even in pre-digested, intact and native form. In this regard, CZE has made a strong contribution in the field of analysis and characterization of monoclonal antibodies (mAb) biotherapeutics, due to its high resolution power and separation mechanism based on differential migration, demonstrating challenging features for discriminating variants with rapid and high-efficiency analyses and for assessment of manufacturing quality control and safety of the final product. This review aims to highlight the most recent applications of CZE-MS in separation and characterization of mAb biotherapeutics based on the most recent literature (2022-2025). The various analytical approaches used in proteins MS characterization will be illustrated, with particular emphasis on the top-down approach, widely applied in this field of analysis and offering comprehensive characterization of proteoforms and exploration of protein interactions under native conditions. CZE-MS demonstrated an effective analytical strategy for the detailed characterization of mAb proteoforms with high potential for application in the challenging task of monitoring mAb release in advanced drug delivery systems.